We recently reported that one important component of virulence in invasive strains of the marine fish pathogen Vibrio anguillarum is a very efficient plasmid-mediated iron sequestering system which enables bacteria to proliferate in the vertebrate host body fluids and tissues in which iron is complexed to iron binding proteins like transferrin and lactoferrin, and thus unabailable for bacterial use. Concomitant with an efficient uptake of iron, two new outer membrane proteins are induced in V. anguillarum under iron deprivation. One of them, OM 2, is induced only in those cells carrying the virulence plasmid pJM1. We propose to characterize the plasmid (and possibly chromosomal) genes involved in the iron uptake mechanism of V. anguillarum. The features of the plasmid-mediated virulence determinant makes the system sifh-V. anguillarum a very attractive model to study host-bacteria interaction. Specifically, we intent to: 1) Analyze the cellular products involved in the mechanisms of iron uptake in V. anguillarum; 2) Generate iron uptake mutants by insertional inactivation with the transposition sequence Tn 5; 3) characterize the OM 2 protein and a shorter polypeptide produced by an iron uptake deficient mutant obtained by Tn 1 insertion on the pJM1 plasmid; 4) Localize the iron uptake genes on the pJM1 plasmid by restriction endonuclease analysis of Tn 5 insertion mutants and molecular cloning. This project will be of the utmost importance in the understanding of a virulence-associated property, iron uptake, which plays an important role in the pathogenesis of many invasive septicemic diseases in man and domestic animals.